Frontiers in Chemistry (Aug 2021)

Wobble Editing of Cre-box by Unspecific CRISPR/Cas9 Causes CCR Release and Phenotypic Changes in Bacillus pumilus

  • Yingxiang Wang,
  • Yingxiang Wang,
  • Linfeng Cao,
  • Linfeng Cao,
  • Meiying Bi,
  • Meiying Bi,
  • Sicheng Wang,
  • Sicheng Wang,
  • Meiting Chen,
  • Meiting Chen,
  • Xingyu Chen,
  • Xingyu Chen,
  • Ming Ying,
  • Ming Ying,
  • Lei Huang,
  • Lei Huang

DOI
https://doi.org/10.3389/fchem.2021.717609
Journal volume & issue
Vol. 9

Abstract

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CRISPR-associated Cas9 endonuclease (CRISPR/Cas9) systems are widely used to introduce precise mutations, such as knocking in/out at targeted genomic sites. Herein, we successfully disrupted the transcription of multiple genes in Bacillus pumilus LG3145 using a series of unspecific guide RNAs (gRNAs) and UgRNA:Cas9 system-assisted cre-box editing. The bases used as gRNAs shared 30–70% similarity with a consensus sequence, a cis-acting element (cre-box) mediating carbon catabolite repression (CCR) of many genes in Bacillus. This triggers trans-crRNA:Cas9 complex wobble cleavage up/downstream of cre sites in the promoters of multiple genes (up to 7), as confirmed by Sanger sequencing and next-generation sequencing (NGS). LG3145 displayed an obvious CCR release phenotype, including numerous secondary metabolites released into the culture broth, ∼ 1.67 g/L white flocculent protein, pigment overflow causing orange-coloured broth (absorbance = 309 nm), polysaccharide capsules appearing outside cells, improved sugar tolerance, and a two-fold increase in cell density. We assessed the relationship between carbon catabolite pathways and phenotype changes caused by unspecific UgRNA-directed cre site wobble editing. We propose a novel strategy for editing consensus targets at operator sequences that mediates transcriptional regulation in bacteria.

Keywords